Parallel functional testing identifies enhancers active in early postnatal mouse brain

Lambert, Jason T.; Su-Feher, Linda; Cichewicz, Karol; Warren, Tracy L.; Zdilar, Iva; Wang, Yurong; Lim, Kenneth J.; Haigh, Jessica L; Morse, Sarah; Canales, Cesar P; Stradleigh, Tyler W.; Palacios, Erika Castillo; Haghani, Viktoria; Moss, Spencer D; Parolini, Hannah; Quintero, Diana; Shrestha, Diwash; Vogt, Daniel; Byrne, Leah C.; Nord, A.

doi:10.7554/elife.69479

Cited by 29 publications

(25 citation statements)

References 89 publications

(111 reference statements)

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“…One potential avenue for testing putative human-specific regulatory elements could be via the introduction of these sequences in mice by CRISPR-mediated gene editing or massive parallel reporter assay to determine whether GATA3 expression is subsequently abolished from IHCs in adult mice cochleae (Lambert et al, 2021; K. Li et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Identification of putative GATA3 regulatory elements and comparison of GATA3 distribution in cochleae of mice, rats, macaques, and humans

Ghosh

Wineski

López

et al. 2022

Preprint

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The transcription factor GATA3 plays a critical role in the development of neurons and sensory epithelia of the inner ear. In mouse cochleae, GATA3 is downregulated in certain supporting cells (SCs) and in type I spiral ganglion neurons (SGNs) after development. This reduction of GATA3 in SCs severely limits Atoh1-induced hair cell (HC) regeneration and suggests that a similar downregulation in human cochleae may be limiting for regenerative therapies. However, it is unknown whether GATA3 is similarly or differentially regulated in primates versus rodents. Using CAGE-seq data, we compared over 40 putative GATA3 regulatory elements across species and found both conserved and non-conserved sequences. To assess whether cochlear GATA3 distribution is similar or different between rodents and primates, we immunostained cochleae from mice, rats, macaques, and humans using antibodies raised against highly conserved GATA3 peptide sequences. GATA3 immunostaining in the organs of Corti from all four species revealed a large degree of conservation, where SCs medial and lateral to cochlear HCs exhibited robust nuclear GATA3 immunolabeling, but pillar and Deiters cells had significantly reduced GATA3 immunoreactivity. In all four species, GATA3 was expressed in a subset of SGNs that largely co-expressed peripherin suggesting they were type II SGNs. Only one difference emerged, wherein human cochlear inner hair cells were not GATA3 immunoreactive despite being so in the other species. Overall, the pattern of GATA3 expression in primates appears similar to rodents and reinforces the notion that ATOH1 mediated regenerative therapies may be limited by reduced GATA3 expression in adult SCs.

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Section: Discussionmentioning

confidence: 99%

Identification of putative GATA3 regulatory elements and comparison of GATA3 distribution in cochleae of mice, rats, macaques, and humans

Ghosh

Wineski

López

et al. 2022

Preprint

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“…Thus, expression abundance of each element is a quantifiable measure of its regulatory strength and can be traced down by barcoding ( Arnold et al, 2013 ). STARR-seq was recently used to screen a library of candidate brain-specific enhancers via recombinant adeno-associated virus delivery to early postnatal mouse brains ( Lambert et al, 2021 ).…”

Section: Enhancersmentioning

confidence: 99%

Epigenetics of neural differentiation: Spotlight on enhancers

Giacoman-Lozano

Meléndez-Ramírez

Martínez-Ledesma

et al. 2022

Front. Cell Dev. Biol.

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Neural induction, both in vivo and in vitro, includes cellular and molecular changes that result in phenotypic specialization related to specific transcriptional patterns. These changes are achieved through the implementation of complex gene regulatory networks. Furthermore, these regulatory networks are influenced by epigenetic mechanisms that drive cell heterogeneity and cell-type specificity, in a controlled and complex manner. Epigenetic marks, such as DNA methylation and histone residue modifications, are highly dynamic and stage-specific during neurogenesis. Genome-wide assessment of these modifications has allowed the identification of distinct non-coding regulatory regions involved in neural cell differentiation, maturation, and plasticity. Enhancers are short DNA regulatory regions that bind transcription factors (TFs) and interact with gene promoters to increase transcriptional activity. They are of special interest in neuroscience because they are enriched in neurons and underlie the cell-type-specificity and dynamic gene expression profiles. Classification of the full epigenomic landscape of neural subtypes is important to better understand gene regulation in brain health and during diseases. Advances in novel next-generation high-throughput sequencing technologies, genome editing, Genome-wide association studies (GWAS), stem cell differentiation, and brain organoids are allowing researchers to study brain development and neurodegenerative diseases with an unprecedented resolution. Herein, we describe important epigenetic mechanisms related to neurogenesis in mammals. We focus on the potential roles of neural enhancers in neurogenesis, cell-fate commitment, and neuronal plasticity. We review recent findings on epigenetic regulatory mechanisms involved in neurogenesis and discuss how sequence variations within enhancers may be associated with genetic risk for neurological and psychiatric disorders.

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“…MPRAs have also been adapted to study gene regulation in the brain in vivo, but the current assays lack the sensitivity to detect subtle differences in activity from disrupting individual transcription factor binding sites or SNPs. Both electroporation and adeno-associated viruses (AAVs) have been used to deliver MPRA libraries to the mouse brain 23,[32][33][34] . Although these studies have the sensitivity to measure the tissue or even cell type-specificity of enhancers 35 , limitations in the number of cells receiving the libraries has made it impossible to detect the impact of genetic variants on brain gene regulation 33,36 .…”

Section: Main Textmentioning

confidence: 99%

Anin vivomassively parallel platform for deciphering tissue-specific regulatory function

Brown

Fox

Kaplow

et al. 2022

Preprint

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Genetic studies are rapidly identifying non-protein-coding human disease-associated loci. Understanding the regulatory mechanisms underlying these loci remains a challenge because the causal variants and the tissues in which they act are often unclear. Massively parallel reporter assays (MPRAs) have the potential to link differences in genome sequence, including genetic variants, to tissue-specific regulatory function. Although MPRA and similar technologies have been widely adopted in cell culture, there have been several barriers to widespread use in animals. We overcome these challenges with a new whole-animal MPRA (WhAMPRA), where systemic intravenous AAV effectively transduces the plasmid MPRA library to mouse tissues. Our WhAMPRA approach revealed models of tissue-specific regulation that generally match machine learning model predictions. In addition, we measured the regulatory effects of disrupting MEF2C transcription factor binding sites and impacts of late onset Alzheimers disease-associated genetic variations. Overall, our WhAMPRA technology simultaneously determines the transcriptional functions of hundreds of enhancers in vivo across multiple tissues.

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Parallel functional testing identifies enhancers active in early postnatal mouse brain

Cited by 29 publications

References 89 publications

Identification of putative GATA3 regulatory elements and comparison of GATA3 distribution in cochleae of mice, rats, macaques, and humans

Identification of putative GATA3 regulatory elements and comparison of GATA3 distribution in cochleae of mice, rats, macaques, and humans

Epigenetics of neural differentiation: Spotlight on enhancers

Anin vivomassively parallel platform for deciphering tissue-specific regulatory function

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